The fused anthranilate synthase from Streptomyces venezuelae functions as a monomer
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Recently, we showed that the fused chorismate-utilizing enzyme from the antibiotic-producing soil bacterium Streptomyces venezuelae is an anthranilate synthase (designated SvAS), not a 2-amino-2-deoxyisochorismate (ADIC) synthase, as was predicted based on its amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE (an ADIC synthase). Here, we report the characterization of SvAS using steady-state kinetics, gel filtration chromatography, and laser light scattering. The recombinant His-tagged enzyme has Michaelis constants Km with respect to substrates chorismate and glutamine of 8.2 ± 0.2 μM and 0.84 ± 0.05 mM, respectively, and a catalytic rate constant k cat of 0.57 ± 0.02 s−1 at 30 °C. Unlike most other anthranilate synthases, SvAS does not utilize ammonia as a substrate. The enzyme is competitively but non-cooperatively inhibited by tryptophan (K i = 11.1 ± 0.1 μM) and is active as a monomer. The finding that SvAS is a monomer jibes with the variety of association modes that have been observed for anthranilate synthases from different microorganisms, and it identifies the enzyme’s minimal functional unit as a single TrpE–TrpG pair.
KeywordsAnthranilate synthase Chorismate-utilizing enzyme Fused enzyme Streptomyces venezuelae
We acknowledge support from the U. S. National Institutes of Health (NIH) RCMI (Grant number 2G12RR003048-18) and MBRS-SCORE programs (Grants number 3S06GM0816-33S1 and 1SC3GM083752) to WMB. We thank Tin-Wein Yu, formerly of the University of Washington, for assistance with the development of protocols for expression of SvAS, Santiago Ramon-Maiques of the Centro Nacional de Investigaciones Oncológicas in Madrid for helpful discussions about the structure of the enzyme, and Michael J. Eck of Harvard University for providing us with the high resolution images used in Fig. 4. Disclaimer: certain commercial equipment, instruments, and materials are identified in this paper in order to specify the experimental procedure as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the material, instrument, or equipment identified is necessarily the best available for the purpose.
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